Transposable elements (hereinafter "transposons") are natural gene transfer vectors in bacteria, yeast, Drosophila melanogaster and other organisms. The best documented examples of transposons in bacteria are those carrying genes that confer antibiotic resistance on the bacterium in which they reside. These transposons tend to accumulate and become a part of bacterial plasmids. The biological properties of the plasmids permit the spread of the plasmids and their passengers, e.g., drug resistance transposons, in bacterial populations.
In higher organisms, transposons have been, or are being, used in several ways. For example, transponsons are used as mutagens on the Ti plasmid of Agrobacterium tumefaciens. That is, a method for using bacterial transposons to cause insertion mutuations in the Agrobacterium tumefaciens Ti plasmid, the causative agent of crown gall disease in dicotyledonous plants, has been developed. (See Zambriski, P., Goodman, H., Van Montagu, M. and Schell, J., Mobile Genetic Elements, J. Shapiro, Ed., (Academic Press) New York, pp. 506-535 (1983)). By this technique, it has become possible to identify the plasmid-borne genes that are responsible for virulence, as well as those that are responsible for the tumorous transformation of plant cells caused by the Ti plasmid. Further, it has become possible to show by using transposable elements, that a portion of the Ti plasmid can be integrated into plant genomes and can act as a vehicle for transferring genes from virtually any organism to any dicotyledonous plant that is susceptible to Agrobacterium tumefaciens.
Transposons have also been used to identify and isolate otherwise inaccessible genes (See Bingham, P. M., Kidwell, M. G. and Rubin, G. M., Cell 29:995-1004 (1982)). That is, the White locus of Drosphilia melanogaster has been isolated by virtue of the existence of an insertion mutution at the locus caused by a transposon that has been isolated and studied using recombinant DNA technology. Such applications are receiving increasing attention in plants and animals.
The use of transposable elements as deliberate gene transfer vectors evolved from work in bacteria and yeast and, as stated above, has recently been developed into a useful research tool in Drosolphila melanogaster (See Rubin, G. M. and Spradling, A. C., Science 218:348-353 (1982)). The basic principle on which such applications are based is that transposons are compact genetic units that contain within their sequences essentially all of the coding information required for transposition. Although the transposition functions are only now beginning to be identified in higher organisms, in bacteria, they are known to include enzymes termed transposases, as well as molecules which regulate expression of the transposase and other genes encoding transposition-specific proteins.
In maize, a monocotyledon, transposable elements were first genetically identified in the mid-1940s. These elements have been studied extensively and their genetic behavior has been extensively reviewed (See McClintock, B., Cold Spring Harbor Symp. Quant. Biol. 16:13-47 (1951); McClintock, B., Cold Spring Harbor Symp. Quant. Biol. 21:197-216 (1956); McClintock, B., Brookhaven Symp. Biol. 18:162-184 (1965); Fincham, J. R. S. , and Sastry, G. R. K., Ann. Rev. Genet. 8:15-50 (1974); and Fedoroff, N., Mobile Genetic Elements, J. Shapiro, Ed., (Academic Press) New York, pp. 1-63 (1983)).
It has been demonstrated that transposons are normal, although cryptic, residents of the maize genome and that upon activation, they are responsible for various types of genetic rearrangements, including chromosome breakage, deletions, duplications, inversions and translocations. In addition, it has been shown that certain common types of unstable mutations, which have been studied for decades in both maize and in other organisms, are attributable to the insertion of transposons into genes or genetic loci.
Currently, there is a great deal of interest in the development of gene transfer vectors for use with agriculturally important plants (See Outlook for Science and Technology, The Next Five Years, Vol. III (National Science Foundation (1982); and O.T.A. Report, Impact of Applied Genetics (1981)).
Although the United States presently has an excess productivity in the agricultural sector, this is recognized as a local and short term condition. Thus, agricultural research and planning must be based on long term considerations. The variety of problems surrounding increases in population, degradation of prime farm land and decreasing availability of good farm land necessitates the increased use of marginal land, as well as exogenous fertilizers and chemical pest control compositions.
Classical plant breeding programs have thus far been successful in increasing agricultural productivity. However, a substantial fraction of the increase in farm productivity experienced in the United States in the past 40 years is attributable to the use of fertilizers and modern energyintensive cultivation practices, both of which are increasingly costly. The ability of plant breeding alone to sustain productivity is a matter of some question. Plant breeders are divided in their views on whether genetic improvements will continue at the rate that has occurred over the past few decades or will begin to level out. Since such questions cannot be resolved a priori, it is prudent to explore a variety of additional means by which agronomically useful traits can be accumulated and improved in major crop plants. The unconventional areas that are presently receiving the most attention in the academic research establishment, as well as in both small and large firms with plant-oriented research programs are wide genetic crosses, tissue culture and the development of gene transfer systems that circumvent fertility barriers.
In the past, many attempts have been made to transfrom plant cells with DNA from a variety of sources. The first unequivocal demonstration that DNA transfer can and does occur in plants emerged from the work described above on Agrobacterium tumefaciens Ti plasmid. However, Ti-plasmid mediated gene transfer is presently accomplished only in dicotyledonous plants that interact with the plasmid's natural host bacterium. Since most major crop species are monocotyledonous, ti-plasmid mediated gene transfer has limited applications.